Following the loss of nigrostriatal dopamine (DA), there is increased activity of glutamate neurons within the subthalamic nucleus. These excitatory neurons project to the internal globus pallidus [GPi, or entopeduncular nucleus (EPN) in the rodent], which utilizes the inhibitory neurotransmitter, GABA (gamma aminobutyric acid). In Parkinson's disease (PD), deep brain stimulation (DBS) of the GPi results in improvement in motor function and provides symptomatic relief. However in a rodent PD model, stimulation of the EPN/GPi did not result in any protection against motor deficits or DA loss following acute intrastriatal infusion of 6-hydroxydopamine. DBS could also be damaging the fibers of passage. However, directly decreasing GABA release from the EPN/GPi neurons without affecting the fibers of passage could answer this concern. Since EPN/GPi neurons utilize the vesicular GABA transporter (VGAT) for uptake of GABA into synaptic vesicles, deletion of this gene would selectively decrease the release of GABA from those EPN/GPi neurons. Using the Cre/loxP recombinase gene technology where a specific gene can be silenced, effecting GABA release from the EPN/GPi GABA neurons, can be achieved through deleting a targeted gene in the specific brain area by injecting AAV-Cre into mice that are floxed for the GABA transporter, VGAT (Vgatflox/flox). To determine if deletion of the Vgat gene in the GPi/EPN can be neuroprotective against DA terminal and cell loss using the neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we find that unilateral infusion of AAV-Cre-GFP (green fluorescent protein) into the EPN/GPi labeled approximately 80-90% of those GABA neurons, as determined by GFP staining of EPN/GPi neurons, and increased GABA immuno-gold labeling within the terminals of the motor thalamus (i.e., the EPN/GPi projects to the motor thalamus). As measured by tyrosine hydroxylase (TH) immunoreactivity, this resulted in a bilateral protection from the loss of DA terminals in the striatum and improved motor function. There was partial protection (~50%) from TH/DA neuron loss in the substantia nigra pars compacta (SNpc). Following neurointervention, with unilateral AAV- cre-GFP infusion into the EPN/GPi followed immediately by MPTPtreatment (MPTP/Cre), we find that there is improved motor strength and increased TH protein expression in the striatum and SNpc in the MPTP/Cre vs MPTP only group. Using an additional PD animal model, in which AAV-alpha synuclein (A-Syn) is bilaterally infused into the SNpc, prior deletion of the Vgat gene in the EPN/GPi with AAV-Cre (i.e., protection) resulted in improved motor strength and blockade of TH/DA cell loss in the SNpc compared to the A-Syn only group. The overall goal of this project is to determine whether unilateral deletion of the Vgat gene, in 2 animal models of PD, within the EPN/GPi, following (i.e., neurorestoration, a more translationally relevant model) either progressive administration of MPTP or intranigral infusion of A-Syn, can bilaterally reverse the loss of DA within the nigrostriatal pathway in both young and aged mice. An additional goal is to determine the role of glutamate in the striatum in reversing the DA depletion due to MPTP. The specific aims of this proposal are to: 1.) determine if unilateral deletion of the Vgat gene within the EPN/GPi initiated 4 weeks after progressive MPTP administration can bilaterally reverse the loss of DA markers in the striatum/SN and improve motor function in both young and aged mice, 2.) determine if unilateral deletion of the Vgat gene within the EPN/GPi initiated 8 weeks following bilateral infusion of AAV-A-Syn (mutant form: A53T) into the substantia nigra pars compacta can bilaterally reverse the loss of DA markers in the striatum/SN and improve motor function in both young and aged mice and 3.) determine if the mechanism behind the striatal TH/DA restoration in the MPTP-treated mice following deletion of the Vgat gene within the EPN/GPi is due to increased striatal glutamate. Glutamate antagonists will be infused into the striatum to determine if this will block the DA recovery due to deletion of the Vgat gene in the EPN/GPi following MPTP.